EvdS6 is a bifunctional decarboxylase from the everninomicin gene cluster.

The everninomicins are bacterially produced antibiotic octasaccharides characterized by the presence of two interglycosidic spirocyclic ortho-δ-lactone (orthoester) moieties. The terminating G- and H-ring sugars, L-lyxose and C-4 branched sugar ß-D-eurekanate, are proposed to be biosynthetically derived from nucleotide diphosphate pentose sugar pyranosides; however, the identity of these precursors and their biosynthetic origin remain to be determined. Herein we identify a new glucuronic acid decarboxylase from Micromonospora belonging to the superfamily of short-chain dehydrogenase/reductase enzymes, EvdS6. Biochemical characterization demonstrated that EvdS6 is an NAD+-dependent bifunctional enzyme that produces a mixture of two products, differing in the sugar C-4 oxidation state. This product distribution is atypical for glucuronic acid decarboxylating enzymes, most of which favor production of the reduced sugar and a minority of which favor release of the oxidized product. Spectroscopic and stereochemical analysis of reaction products revealed that the first product released is the oxidatively produced 4-keto-D-xylose and the second product is the reduced D-xylose. X-ray crystallographic analysis of EvdS6 at 1.51 Å resolution with bound co-factor and TDP demonstrated that the overall geometry of the EvdS6 active site is conserved with other SDR enzymes and enabled studies probing structural determinants for the reductive half of the net neutral catalytic cycle. Critical active site threonine and aspartate residues were unambiguously identified as essential in the reductive step of the reaction and resulted in enzyme variants producing almost exclusively the keto sugar. This work defines potential precursors for the G-ring L-lyxose and resolves likely origins of the H-ring ß-D-eurekanate sugar precursor.

An intricate regulation of WblA controlling production of silent tylosin analogues and abolishment of expressible nikkomycin.

Genome sequencing has revealed that actinomycetes possess the potential to produce many more secondary metabolites than previously thought. The existing challenge is to devise efficient methods to activate these silent biosynthetic gene clusters (BGCs). In Streptomyces ansochromogenes, disruption of wblA, a pleiotropic regulatory gene, activated the expression of cryptic tylosin analogues and abolished nikkomycin production simultaneously. Overexpressing pathway-specific regulatory genes tylR1 and tylR2 can also trigger the biosynthesis of silent tylosin analogues, in which TylR1 exerted its function via enhancing tylR2 expression. Bacterial one-hybrid system experiments unveiled that WblA directly inhibits the transcription of tylR1 and tylR2 to result in the silence of tylosin analogues BGC. Furthermore, WblA can activate the nikkomycin production through up-regulating the transcription of pleiotropic regulatory gene adpA. More interestingly, AdpA can activate sanG (an activator gene in nikkomycin BGC) but repress wblA. Our studies provide a valuable insight into the complex functions of pleiotropic regulators.

Resistance traits and molecular characterization of multidrug-resistant Acinetobacter baumannii isolates from an intensive care unit of a tertiary hospital in Guangdong, southern China.

PURPOSE: This study aims to characterize antimicrobial resistance (AMR) of all the non-duplicated Acinetobacter baumannii strains isolated from an intensive care unit in a tertiary hospital during the period of January 1 to December 31, 2015. METHODS: A. baumannii (n = 95 strains) isolated from patients was subjected to antimicrobial susceptibility test (AST) by Vitek 2 Compact system to determine minimum inhibitory concentrations, followed by genotyping by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). Resistance genes of interest were PCR amplified and sequenced. RESULTS: All isolates were qualified as MDR, with a resistance rate of > 80% to 8 antimicrobials tested. In terms of beta-lactamase detection, the blaOXA23, blaTEM-1, and armA genes were detected frequently at 92.63%, 9 1.58%, and 88.42%, respectively. The metallo-ß-lactamase genes blaIMP and blaVIM were undetected. Aph (3')-I was detected in 82 isolates (86.32%), making it the most prevalent aminoglycoside-modifying enzyme (AMEs) encoding gene. In addition, ant (3″)-I was detected at 30.53%, while 26.32% of the strains harbored an aac (6')-Ib gene. ERIC-PCR typing suggested moderate genetic diversity among the isolates, which might be organized into 10 distinct clusters, with cluster A (n = 86 isolates or 90.53%) being the dominant cluster. CONCLUSIONS: All of the A. baumannii strains detected in the ICU were MDR clones exhibiting extremely high resistance to carbapenems and aminoglycosides as monitored throughout the study period. They principally belonged to a single cluster of isolates carrying blaOXA23 and armA co-producing different AMEs genes.

Deficiency in cytosine DNA methylation leads to high chaperonin expression and tolerance to aminoglycosides in Vibrio cholerae.

Antibiotic resistance has become a major global issue. Understanding the molecular mechanisms underlying microbial adaptation to antibiotics is of keen importance to fight Antimicrobial Resistance (AMR). Aminoglycosides are a class of antibiotics that target the small subunit of the bacterial ribosome, disrupting translational fidelity and increasing the levels of misfolded proteins in the cell. In this work, we investigated the role of VchM, a DNA methyltransferase, in the response of the human pathogen Vibrio cholerae to aminoglycosides. VchM is a V. cholerae specific orphan m5C DNA methyltransferase that generates cytosine methylation at 5'-RCCGGY-3' motifs. We show that deletion of vchM, although causing a growth defect in absence of stress, allows V. cholerae cells to cope with aminoglycoside stress at both sub-lethal and lethal concentrations of these antibiotics. Through transcriptomic and genetic approaches, we show that groESL-2 (a specific set of chaperonin-encoding genes located on the second chromosome of V. cholerae), are upregulated in cells lacking vchM and are needed for the tolerance of vchM mutant to lethal aminoglycoside treatment, likely by fighting aminoglycoside-induced misfolded proteins. Interestingly, preventing VchM methylation of the four RCCGGY sites located in groESL-2 region, leads to a higher expression of these genes in WT cells, showing that the expression of these chaperonins is modulated in V. cholerae by DNA methylation.

Altering mammalian transcription networking with ADAADi: An inhibitor of ATP-dependent chromatin remodeling.

Active DNA-dependent ATPase A Domain inhibitor (ADAADi) is the only known inhibitor of ATP-dependent chromatin remodeling proteins that targets the ATPase domain of these proteins. The molecule is synthesized by aminoglycoside phosphotransferase enzyme in the presence of aminoglycosides. ADAADi interacts with ATP-dependent chromatin remodeling proteins through motif Ia present in the conserved helicase domain, and thus, can potentially inhibit all members of this family of proteins. We show that mammalian cells are sensitive to ADAADi but with variable responses in different cell lines. ADAADi can be generated from a wide variety of aminoglycosides; however, cells showed differential response to ADAADi generated from various aminoglycosides. Using HeLa and DU145 cells as model system we have explored the effect of ADAADi on cellular functions. We show that the transcriptional network of a cell type is altered when treated with sub-lethal concentration of ADAADi. Although ADAADi has no known effects on DNA chemical and structural integrity, expression of DNA-damage response genes was altered. The transcripts encoding for the pro-apoptotic proteins were found to be upregulated while the anti-apoptotic genes were found to be downregulated. This was accompanied by increased apoptosis leading us to hypothesize that the ADAADi treatment promotes apoptotic-type of cell death by upregulating the transcription of pro-apoptotic genes. ADAADi also inhibited migration of cells as well as their colony forming ability leading us to conclude that the compound has effective anti-tumor properties.

Aminoglycoside riboswitch control of the expression of integron associated aminoglycoside resistance adenyltransferases.

The proliferation of antibiotic resistance has its origins in horizontal gene transfer. The class 1 integrons mediate gene transfer by assimilating antibiotic-resistance genes through site-specific recombination. For the class 1 integrons the first assimilated gene normally encodes an aminoglycoside antibiotic resistance protein which is either an aminoglycoside acetyltransferase (AAC), nucleotidyltransferase - (ANT), or adenyl transferase (AAD). An aminoglycoside-sensing riboswitch RNA in the leader RNA of AAC/AAD that controls the expression of aminoglycoside resistance genes has been previously described. Here we explore the relationship between the recombinant products of integron recombination and a series of candidate riboswitch RNAs in the 5' UTR of aad (aminoglycoside adenyltransferases) genes. The RNA sequences from the 5' UTR of the aad genes from pathogenic strains that are the products of site-specific DNA recombination by class 1 integrons were investigated. Reporter assays, MicroScale Thermophoresis (MST) and covariance analysis revealed that a functional aminoglycoside-sensing riboswitch was selected at the DNA level through integron-mediated site-specific recombination. This study explains the close association between integron recombination and the aminoglycoside-sensing riboswitch RNA.

An albumin­binding domain and targeting peptide­based recombinant protein and its enediyne­integrated analogue exhibit directional delivery and potent inhibitory activity on pancreatic cancer with K­ras mutation.

Efficient enrichment and transmembrane transport of cytotoxic reagents are considered to be effective strategies to increase the efficiency and selectivity of antitumor drugs targeting solid tumors. In the present study, a recombinant protein ABD­LDP­Ec consisting of the albumin­binding domain (ABD), the apoprotein (LDP) of lidamycin (LDM) and an EGFR­targeting oligopeptide (Ec) was prepared by DNA recombination and bacterial fermentation, and was integrated with the enediyne chromophore (AE) of lidamycin to generate its enediyne­integrated analogue ABD­LDP­Ec­AE. ABD­LDP­Ec exhibited high binding capacity to both albumin and EGFR­positive pancreatic cancer cells, and was internalized into the cytoplasm through receptor­mediated endocytosis and albumin­driven macropinocytosis of K­ras mutant cells. In animal experiments, ABD­LDP­Ec demonstrated notable selective distribution in pancreatic carcinoma xenografts by passive targeting of albumin captured in the blood and was retained in the tumor for 48 h. ABD­LDP­Ec and ABD­LDP­Ec­AE exhibited inhibitory activity in cell proliferation and AsPC­1 xenograft growth, and ABD­LDP­Ec­AE increased the tumor growth inhibition rate by 20% compared with natural LDM. The results indicated that the introduction of ABD­based multi­functional drug delivery may be an effective approach to improve the efficacy of antitumor drugs, especially for K­ras mutant cancers.

Antibiotic Resistance Genes in the Gut Microbiota of Children with Autistic Spectrum Disorder as Possible Predictors of the Disease.

The gut microbiota (GM), which contains thousands of bacterial species, is a reservoir of antibiotic resistance genes (ARGs) called resistome. Early life exposure to antibiotics alters significantly the composition and function of the gut microbiota of children, which may trigger symptoms of autism spectrum disorder (ASD). This is because the GM plays an important role in the bidirectional communication between the gut and the brain and influences the brain normal functioning through multiple pathways. The goal of this article is to study the distribution of ARGs in the GM of 3- to 5-year-old healthy children and children with ASD living in Moscow, Russia. The metagenomic analysis of samples from both groups revealed differences in the signatures between them. The signatures consisted of the bacterial genera and aminoglycoside, ß-lactam, macrolide, and tetracycline resistance genes that they harbored. Our results show an increase in ARGs in the resistome of the GM of children with ASD. These findings emphasize the negative influence of early-life antibiotic therapy. We found three ARGs, aac(6')-aph(2''), cepA-49, and tet(40), which could serve as markers of ASD. The additional functions carried out by the enzymes, encoded by these genes, are being discussed.

High prevalence of carbapenemase, AmpC ß-lactamase and aminoglycoside resistance genes in extended-spectrum ß-lactamase-positive uropathogens from Northern India.

OBJECTIVES: This study investigated the occurrence of extended-spectrum ß-lactamase (ESBL) genes coexisting with carbapenemase, AmpC and aminoglycoside resistance gene in uropathogens in India. METHODS: Antimicrobial susceptibility testing was performed by disk diffusion. Antimicrobial resistance genes were detected by multiplex PCR. RESULTS: Of 1516 consecutive urine samples, 454 (29.9%) showed significant bacteriuria with a single micro-organism, predominantly Escherichia coli (n=343), followed by Klebsiella pneumoniae (n=92), Pseudomonas aeruginosa (n=10) and Proteus mirabilis (n=9). Among the uropathogens, 61 ESBL-producers were identified containing blaCTX-M-15 (n=32), blaCTX-M-15+blaOXA-2 (n=15), blaCTX-M-15+blaOXA-2+blaTEM-1 (n=6), blaOXA-2 (n=5), blaOXA-2+blaSHV-76 (n=1), blaTEM-1+blaSHV-76 (n=1) and blaTEM-1 (n=1). All ESBL genes were located on horizontally transferable plasmids of incompatibility types HI1, I1, FIA+FIB, FIA and Y. Among the 61 ESBL-producers, 59 harboured carbapenemase genes, including blaNDM-5 (n=48), blaNDM-5+blaOXA-48 (n=5), blaNDM-5+blaIMP (n=5) and blaNDM-5+blaIMP+blaVIM (n=1). ESBL-producing uropathogens also harboured 16S rRNA methylase genes, including rmtB (n=9), rmtA (n=4), rmtC (n=1) and armA (n=1). ESBL-positive isolates also contained AmpC genes, including blaCIT (n=8) and blaDHA-1 (n=1). Imipenem and gentamicin had the lowest resistance rates against the uropathogens. CONCLUSION: This is the first report showing the high prevalence of carbapenemases in ESBL-positive isolates in this area. Regular surveillance for such resistance mechanisms will be useful for health personnel to treat infections by these multidrug-resistant pathogens.

Investigation of carbapenemases and aminoglycoside modifying enzymes of Acinetobacter baumannii isolates recovered from patients admitted to intensive care units in a tertiary-care hospital in Brazil

Abstract INTRODUCTION: Acinetobacter baumannii are opportunistic bacteria, highly capable of acquiring antimicrobial resistance through the production of carbapenemases and aminoglycoside modifying enzymes (AMEs). METHODS: Carbapenemase and AME genes were investigated in A. baumannii recovered from inpatients of a Brazilian hospital. RESULTS: The key genes found were bla OXA-51-like, the association ISAba1- bla OXA-23-like, and the AME genes aph(3´)-VI, aac(6´)-Ib, aac(3)-Ia, and aph(3´)-Ia. Different clusters spread through the institution wards. CONCLUSIONS: The dissemination of bla OXA-23-like and AME-carrying A. baumannii through the hospital highlights the need for improved preventive measures to reduce the spread of infection.

Investigation of carbapenemases and aminoglycoside modifying enzymes of Acinetobacter baumannii isolates recovered from patients admitted to intensive care units in a tertiary-care hospital in Brazil.

INTRODUCTION: Acinetobacter baumannii are opportunistic bacteria, highly capable of acquiring antimicrobial resistance through the production of carbapenemases and aminoglycoside modifying enzymes (AMEs). METHODS: Carbapenemase and AME genes were investigated in A. baumannii recovered from inpatients of a Brazilian hospital. RESULTS: The key genes found were bla OXA-51-like, the association ISAba1- bla OXA-23-like, and the AME genes aph(3´)-VI, aac(6´)-Ib, aac(3)-Ia, and aph(3´)-Ia. Different clusters spread through the institution wards. CONCLUSIONS: The dissemination of bla OXA-23-like and AME-carrying A. baumannii through the hospital highlights the need for improved preventive measures to reduce the spread of infection.

Assessment of antibiotic resistance in bacteriophage-insensitive Klebsiella pneumoniae.

This study was design to evaluate the physiological properties of bacteriophage-insensitive Klebsiella pneumoniae (BIKP) mutants in association with the antibiotic cross-resistance, ß-lactamase activity, and gene expression. Klebsiella pneumoniae ATCC 23357(KPWT), ciprofloxacin-induced antibiotic-resistant K. pneumoniae ATCC 23357 (KPCIP), and clinically isolated antibiotic-resistant K. pneumoniae 10263 (KPCLI) were used to isolate BIKP mutants against KPB1, PBKP02, PBKP21, PBKP29, PBKP33, and PBKP35. PBKP35-induced mutants, including bacteriophage-insensitive K. pneumoniae ATCC 23357 (BIKPWT), ciprofloxacin-induced K. pneumoniae ATCC 23357 (BIKPCIP), and clinically isolated antibiotic-resistant K. pneumoniae CCARM 10263 (BIKPCLI). BIKPWT, BIKPCIP, and BIKPCLI were resistant to Klebsiella bacteriophages, KPB1, PBKP02, PBKP21, PBKP29, and PBKP33. The antibiotic cross-resistance to cefotaxime, cephalothin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, levofloxacin, and nalidixic acid was observed in BIKPWT. The relative expression levels of vagC was increased by more than 8-folds in BIKPWT, corresponding to the increased ß-lactamase activity. The aac(6')-Ib-cr was overexpressed in BIKP mutants, responsible for aminoglycoside and quinolone resistance. The phage-resistant mutants decreased the antibiotic susceptibilities in association with ß-lactamase activity and antibiotic resistance-related gene expression. The results pointed out the cross-resistance of BIKP mutants to antibiotics, which might be considered when applying for the therapeutic use of bacteriophage.

Emergence of aph(3')-VI and accumulation of aminoglycoside modifying enzyme genes in KPC-2-possessing Enterobacter aerogenes isolates from infections and colonization in patients from Recife-PE, Brazil.

INTRODUCTION: The objective of this study was to characterize genes of aminoglycoside modifying enzymes (AMEs) in colonizing and infecting isolates of E. aerogenes harboring bla KPC from patients at a public hospital in Recife-PE, Brazil. METHODS: We analyzed 29 E. aerogenes clinical isolates resistant to aminoglycosides. AMEs genes were investigated by PCR and sequencing. RESULTS: Colonizing and infecting isolates mainly presented the genetic profiles aac(3)-IIa/aph(3')-VI or ant(2")-IIa/aph(3')-VI. This is the first report of aph(3')-VI in E. aerogenes harboring bla KPC in Brazil. CONCLUSIONS: The results highlight the importance in establishing rigorous methods for the surveillance of resistance genes, especially in colonized patients.

Integron-Derived Aminoglycoside-Sensing Riboswitches Control Aminoglycoside Acetyltransferase Resistance Gene Expression.

Class 1 integrons accumulate antibiotic resistance genes by site-specific recombination at aatI-1 sites. Captured genes are transcribed from a promoter located within the integron; for class 1 integrons, the first gene to be transcribed and translated normally encodes an aminoglycoside antibiotic resistance protein (either an acetyltransferase [AAC] or adenyltransferase [AAD]). The leader RNA from the Pseudomonas fluorescens class 1 integron contains an aminoglycoside-sensing riboswitch RNA that controls the expression of the downstream aminoglycoside resistance gene. Here, we explore the relationship between integron-dependent DNA recombination and potential aminoglycoside-sensing riboswitch products of recombination derived from a series of aminoglycoside-resistant clinical strains. Sequence analysis of the clinical strains identified a series of sequence variants that were associated with class I integron-derived aminoglycoside-resistant (both aac and aad) recombinants. For the aac recombinants, representative sequences showed up to 6-fold aminoglycoside-dependent regulation of reporter gene expression. Microscale thermophoresis (MST) confirmed RNA binding. Covariance analysis generated a secondary-structure model for the RNA that is an independent verification of previous models that were derived from mutagenesis and chemical probing data and that was similar to that of the P. fluorescens riboswitch RNA. The aminoglycosides were among the first antibiotics to be used clinically, and the data suggest that in an aminoglycoside-rich environment, functional riboswitch recombinants were selected during integron-mediated recombination to regulate aminoglycoside resistance. The incorporation of a functional aminoglycoside-sensing riboswitch by integron recombination confers a selective advantage for the expression of resistance genes of diverse origins.

Development of a Luminex xTAG Assay for the Rapid Detection of Five Aminoglycoside Resistance Genes Both in Staphylococci and Enterococci.

Resistance to aminoglycoside antibiotics is now common in pathogenic bacteria, making treatment of infections difficult. The rapid spread of resistance is mainly related to the dissemination of genes encoding aminoglycoside-modifying enzymes (AMEs). Staphylococci and enterococci are opportunistic human pathogens capable of causing a wide range of infections. Isolates from clinical cases are often found to be resistant to aminoglycosides. The aim of the present study was to develop a bead-based xTAG assay for the simultaneous detection of five prevalent aminoglycoside resistance genes in staphylococci and enterococci, including aac(6')-Ie-aph(2″)-Ia, aph(3')-IIIa, ant(4')-Ia, ant(9)-Ia, and ant(6)-Ia. The limit of detection ranged from 10 to 1000 copies/µL of input purified plasmid DNA. Twenty-two bacterial isolates from clinical samples were examined using the newly developed xTAG assay and also by conventional PCR to determine the relative performance of each. The results obtained by xTAG assay showed higher detection rates and accuracy for AME genes than conventional PCR. It indicated that the xTAG-multiplex PCR method is a high-throughput tool for rapid identification of AME genes.

C-Nucleoside Formation in the Biosynthesis of the Antifungal Malayamycin A.

Malayamycin A is an unusual bicyclic C-nucleoside, with interesting antiviral, antifungal, and anticancer bioactivity. We report here the discovery and characterization of the biosynthetic pathway to malayamycin by using genome mining of near-identical clusters both from the known producer Streptomyces malaysiensis and from Streptomyces chromofuscus. The key precursor 5'-pseudouridine monophosphate (5'-Ψ-MP) is supplied chiefly through the action of MalD, a TruD-like pseudouridine synthase. In vitro assays showed that MalO is an enoylpyruvyltransferase acting almost exclusively on 5'-Ψ-MP rather than 5'-UMP, while in contrast the counterpart enzyme NikO in the nikkomycin pathway readily accepts either substrate. As a result, deletion of malD in S. chromofuscus coupled with introduction of the gene for NikO led to production of non-natural N-malayamycin, as well as malayamycin A. Conversely, cloning malO into the nikkomycin producer Streptomyces tendae in place of nikO diverted biosynthesis toward C-nucleoside formation.

In Vivo Characterization of Phosphotransferase-Encoding Genes istP and forP as Interchangeable Launchers of the C3',4'-Dideoxygenation Biosynthetic Pathway of 1,4-Diaminocyclitol Antibiotics.

Deactivation of aminoglycosides by their modifying enzymes, including a number of aminoglycoside O-phosphotransferases, is the most ubiquitous resistance mechanism in aminoglycoside-resistant pathogens. Nonetheless, in a couple of biosynthetic pathways for gentamicins, fortimicins, and istamycins, phosphorylation of aminoglycosides seems to be a unique and initial step for the creation of a natural defensive structural feature such as a 3',4'- dideoxy scaffold. Our aim was to elucidate the biochemical details on the beginning of these C3',4'-dideoxygenation biosynthetic steps for aminoglycosides. The biosynthesis of istamycins must surely involve these 3',4'-didehydroxylation steps, but much less has been reported in terms of characterization of istamycin biosynthetic genes, especially about the phosphotransferase-encoding gene. In the disruption and complementation experiments pointing to a putative gene, istP, in the genome of wild-type Streptomyces tenjimariensis, the function of the istP gene was proved here to be a phosphotransferase. Next, an in-frame deletion of a known phosphotransferase-encoding gene forP from the genome of wild-type Micromonospora olivasterospora resulted in the appearance of a hitherto unidentified fortimicin shunt product, namely 3-O-methyl-FOR-KK1, whereas complementation of forP restored the natural fortimicin metabolite profiles. The bilateral complementation of an istP gene (or forP) in the ΔforP mutant ( or ΔistP mutant strain) successfully restored the biosynthesis of 3',4'- dideoxy fortimicins and istamycins , thus clearly indicating that they are interchangeable launchers of the biosynthesis of 3',4'-dideoxy types of 1,4-diaminocyclitol antibiotics.

Endemicity of the High-Risk Clone Klebsiella pneumoniae ST340 Coproducing QnrB, CTX-M-15, and KPC-2 in a Brazilian Hospital.

The dissemination of multiresistant Klebsiella pneumoniae carbapenemase (KPC)-2-producing Klebsiella pneumoniae isolates belonging to international high-risk clones poses a major health care threat. In this study, 48 nonduplicated, carbapenem-resistant K. pneumoniae isolated from 2011 to 2014 in a tertiary hospital were investigated. The blaKPC-2 gene was the only determinant for carbapenem resistance. The blaCTX-M-15 gene was the main determinant for the production of extended-spectrum beta-lactamase (ESBL), whereas aph(3')-Ia and qnrB were the most common genes associated with resistance to aminoglycosides and quinolones, respectively. Nine different sequence types (STs) were identified. The most common was ST340. Molecular typing by enterobacterial repetitive intergenic consensus-PCR placed 48 strains among 10 different clusters. In the studied hospital, the high-risk clone of KPC-2-producing K. pneumoniae ST340, harboring genes that codify aminoglycoside modifying enzymes, QnrB and CTX-M-15 plus CTXM-2-type ESBLs, is disseminated and acts as a major agent of infections in critically ill patients.

Discovery of 16-Demethylrifamycins by Removing the Predominant Polyketide Biosynthesis Pathway in Micromonospora sp. Strain TP-A0468.

A number of strategies have been developed to mine novel natural products based on biosynthetic gene clusters and there have been dozens of successful cases facilitated by the development of genomic sequencing. During our study on biosynthesis of the antitumor polyketide kosinostatin (KST), we found that the genome of Micromonospora sp. strain TP-A0468, the producer of KST, contains other potential polyketide gene clusters, with no encoded products detected. Deletion of kst cluster led to abolishment of KST and the enrichment of several new compounds, which were isolated and characterized as 16-demethylrifamycins (referred to here as compounds 3 to 6). Transcriptional analysis demonstrated that the expression of the essential genes related to the biosynthesis of compounds 3 to 6 was comparable to the level in the wild-type and in the kst cluster deletion strain. This indicates that the accumulation of these compounds was due to the redirection of metabolic flux rather than transcriptional activation. Genetic disruption, chemical complementation, and bioinformatic analysis revealed that the production of compounds 3 to 6 was accomplished by cross talk between the two distantly placed polyketide gene clusters pks3 and M-rif This finding not only enriches the analogue pool and the biosynthetic diversity of rifamycins but also provides an auxiliary strategy for natural product discovery through genome mining in polyketide-producing microorganisms.IMPORTANCE Natural products are essential in the development of novel clinically used drugs. Discovering new natural products and modifying known compounds are still the two main ways to generate new candidates. Here, we have discovered several rifamycins with varied skeleton structures by redirecting the metabolic flux from the predominant polyketide biosynthetic pathway to the rifamycin pathway in the marine actinomycetes species Micromonospora sp. strain TP-A0468. Rifamycins are indispensable chemotherapeutics in the treatment of various diseases such as tuberculosis, leprosy, and AIDS-related mycobacterial infections. This study exemplifies a useful method for the discovery of cryptic natural products in genome-sequenced microbes. Moreover, the 16-demethylrifamycins and their genetically manipulable producer provide a new opportunity in the construction of novel rifamycin derivates to aid in the defense against the ever-growing drug resistance of Mycobacterium tuberculosis.

Emergence of aph(3')-VI and accumulation of aminoglycoside modifying enzyme genes in KPC-2-possessing Enterobacter aerogenes isolates from infections and colonization in patients from Recife-PE, Brazil

Abstract INTRODUCTION: The objective of this study was to characterize genes of aminoglycoside modifying enzymes (AMEs) in colonizing and infecting isolates of E. aerogenes harboring bla KPC from patients at a public hospital in Recife-PE, Brazil. METHODS: We analyzed 29 E. aerogenes clinical isolates resistant to aminoglycosides. AMEs genes were investigated by PCR and sequencing. RESULTS: Colonizing and infecting isolates mainly presented the genetic profiles aac(3)-IIa/aph(3')-VI or ant(2")-IIa/aph(3')-VI. This is the first report of aph(3')-VI in E. aerogenes harboring bla KPC in Brazil. CONCLUSIONS: The results highlight the importance in establishing rigorous methods for the surveillance of resistance genes, especially in colonized patients.